The invention relates to the field of vibrational imaging analysis, and relates in particular to the field of coherent anti-Stokes Raman scattering analysis.
Coherent anti-stokes Raman scattering (CARS) imaging provides for the analysis of chemical and biological samples by using molecular vibrations as a contrast mechanism. In particular, CARS analysis uses at least two laser fields, a pump electromagnetic field with a center frequency at ωp and a Stokes electromagnetic field with a center frequency at ωs. The pump and Stokes fields interact with a sample and generate a coherent anti-Stokes field having a frequency of ωas=2ωp−ωs in the phase matched direction. When the frequency difference, ωp−ωs, is tuned to be resonant with a given vibrational mode, an enhanced CARS signal is observed at the anti-Stokes frequency ωas.
Unlike fluorescence microscopy, CARS microscopy does not require the use of fluorophores (which may undergo photobleaching), since the imaging relies on vibrational contrast of biological and chemical materials. Further, the coherent nature of CARS microscopy offers significantly higher sensitivity than spontaneous Raman microscopy. This permits the use of lower average excitation powers and/or short data collection times (which is tolerable for biological samples). The fact that ωas>ωp, ωs allows the signal to be detected in the presence of background fluorescence.
For example, U.S. Pat. No. 4,405,237 discloses a coherent anti-Stokes Raman spectroscopic imaging device in which two laser pulse trains of different wavelengths, temporally and spatially overlapped, are used to simultaneously illuminate a sample. The '237 patent discloses a non-collinear geometry of the two laser beams and a detection of the signal beam in the phase matching direction with a two-dimensional detector, which gives the spatial resolution.
A collinear excitation geometry is proposed in U.S. Pat. No. 6,108,081, which discloses a method and apparatus for microscopic vibrational imaging using coherent anti-Stokes Raman scattering in which collinear pump and Stokes beams are focused by a high numerical aperture (NA) objective lens. The nonlinear dependence of the signal on the excitation intensity ensures a small probe volume of the foci, allowing three-dimensional sectioning across a relatively thick sample. The signal beam is detected in the forward direction.
Following the previous non-collinear excitation geometry however, U.S. Pat. No. 6,934,020 discloses a laser microscope in which pump and Stokes beams are delivered from a light emitting system to a beam irradiating mechanism using an optical fiber. The pump and Stokes beams are combined prior to entering a proximal end of the optical fiber, and then separated again into two beams by the beam irradiating mechanism after leaving the distal end of the optical fiber so that each beam may be directed toward each other at a sample. The CARS signal is detected from the sample in the forward direction.
There is also a non-resonant contribution to the CARS signal, however, that does not carry chemically-specific information that can distort and even overwhelm the resonant signal of interest. The CARS background is caused by electronic contributions to the third order nonlinear susceptibility. The non-resonant contribution arises from both the sample of interest as well as of the surrounding isotropic bulk medium (i.e., solvent), and is independent of the Raman shift, ωp−ωs. This non-resonant contribution provides background with no vibrational contrast from which the desired signal must be filtered or somehow distinguished. The presence of this background from the isotropic bulk water has hindered efforts to increase the sensitivity of CARS imaging, particularly in biological applications.
One approach to reducing the non-resonant background field in CARS spectroscopy is to take advantage of the fact that the non-resonant background has different polarization properties than the resonant signal. For example, U.S. Pat. No. 6,798,507 discloses a system in which the pump and Stokes beams are polarized, and a polarization sensitive detector is employed. In high resolution CARS microscopy, however, tightly focused collinear excitation beams are sometimes necessary and it is known that tightly focusing polarized beams will result in polarization scrambling.
U.S. Pat. No. 6,809,814 discloses a system in which a CARS signal is received in the reverse direction (epi-direction) from the sample. The signal generated in the epi-direction typically includes a significantly higher signal to background ratio, but in some applications, the signal generated in the epi-direction is usually much smaller than that generated in the forward direction.
In addition, conventional CARS microscopy systems generally require that a sample be extracted from a subject and examined, typically while positioned on a stage that may be movable in x and y directions. In certain applications, however, it is desirable to perform CARS analysis of a sample in situ. Such samples, however, may not be easily viewed using forward direction CARS.
There is a need, therefore, for a system and method for providing applications of CARS analyses in situ within a human, animal or other subject or sample.